Abstract
Glioblastoma Multiforme (GBM) is a rare and highly aggressive primary brain tumor characterized by accelerated tumorigenicity and high intratumor heterogeneity. Rapid expansion of tumor cell growth generates regions of the GBM tumor that receive sub-optimal levels of oxygen, termed hypoxic niches. Cancer stem cells (CSCs) residing in the GBM hypoxic niche exhibit enhanced chemoradiotherapy resistance, metastatic potential, self-renewal, and cell survival. Thus, developing a therapeutic equipped to target the hypoxic CSC population is imperative. Plasminogen Activator Inhibitor -1 (PAI-1) is highly upregulated in the GBM hypoxic niche and is associated with angiogenesis, metastasis, and a poor prognosis (Sugiura et al., 1999). Recent studies have demonstrated that PAI-1 may also confer a survival advantage to malignant cells by inhibiting programmed cell death or apoptosis (Fang et al., 2011). Downregulation of PAI-1 using small interfering RNAs (siRNAs) activated the Extrinsic Apoptotic Pathway (EAP) in brain microvascular endothelial cells and human cancer cell lines. (Bajou et al., 2008; Fang et al., 2011). Thus, we hypothesized that the generation of a PAI-1 specific knockout using the WT Cas9 system would elucidate the role of PAI-1 in hypoxic LN-229 cells. For therapeutic relevance, LN-229 cells were also subjected to epigenetic repression using the CRISPRi system. Previous studies have examined the effects of modulating PAI-1 using RNA interference (RNAi), however, use of the CRISPRi system for transcriptional repression required investigation. To downregulate PAI-1 expression in vitro, plasmids encoding components of the CRISPRi system were transfected into LN-229 cells, a Grade IV GBM tumor cell line. We observed a non-significant difference between cells treated with the epigenetic repressor dCas9-KRAB and vehicle control. However, cells treated with dCas9-NE exhibited 57% PAI-1 down-regulation. To characterize the implications of a PAI-1 specific KO (P1KO), we performed single cell plating to produce a P1KO cell line. Our results suggest PAI-1 gene removal induces disengagement from the substratum leading to the loss of LN-229 P1KO cells in culture. To examine the correlation between P1KD/P1KO cells and apoptosis, we used a caspase assay. Our results indicate an immense increase in intracellular caspase 3/7 activity following P1KO and P1KD in LN-229 cells. Strikingly, the majority of fluorescent caspase active cells were suspended in the medium. Thereby, suggesting a P1KD/P1KO regulates de-adherence and apoptotic induction. To investigate the correlation between PAI-1 mediated cellular detachment, oxygen tension and apoptosis, we used a trypan blue staining. We demonstrated that adhered hypoxic and normoxic P1KO cells exhibited 99% viability, whereas normoxic and hypoxic detached cells showed 62.4% and 12% viability. Ultimately, PAI-1 repression induces cellular detachment coupled with the onset of apoptosis that is exacerbated in hypoxic conditions. From a molecular standpoint, we have demonstrated that transcriptional repression of PAI-1 leads to the activation of caspases 3/7, indicators of early apoptosis. In the future, PAI-1 repression in conjunction with standard GBM treatments may be used to target and kill highly aggressive cells in the GBM hypoxic niche. Consequently, GBM patients may experience increased relapse-free survival rates, as hypoxic CSCs are major culprits of tumor recurrence and metastasis.